Allergy is an acquired hypersensitivity disorder of the immune system and is triggered by exposure to harmless environmental substances known as allergens. A type I hypersensitivity reaction is characteristic of allergic reactions and results in the production of excessive amounts of IgE antibodies which in turn activate basophils and mast cells causing an inflammatory reaction. The effects may be systemic such as vasodilation, mucus secretion, nerve stimulation and smooth muscle contraction causing an anaphylaxis reaction, or confined to a particular area of the body, for example the respiratory system.
Food allergy is an emerging major public health problem that affects around 6% of school children and approximately 4% of adults and can have severe consequences, including fatal anaphylactic reactions(1). Allergy can therefore have a significant impact on psychosocial aspects of quality of life extending beyond the immediate clinical effects of the patient's allergic condition and the daily activities of families(2). At present the standard of care for this type of allergy includes strict avoidance of the offending allergens and treatment with epinephrine.
Peanut allergy is a Type I hypersensitivity (IgE mediated) immune response to dietary substances from peanuts causing an overreaction of the immune system. The Asthma and Allergy Foundation of America estimates that peanut allergy is the most common cause of food-related death in the USA and has estimated that it affects 0.4-0.6% of the population. Tree nuts such as pecans, pistachios, pine nuts and walnut are also common nut allergens.
To date eleven allergens (Ara h 1 through Ara h 11) have been identified from peanut (Arachis hypogea) and many of them have been sequenced and cloned. Based on the International Union of Immunological Societies (IUIS) nomenclature these allergens include: Ara h 1, a Cupin (Vicillin-type, 7S globulin) of 64 kDa; Ara h 2, a Conglutin (2S albumin) of 17 kDa; Ara h 3, a Cupin (Legumin-type, 11S globulin, Glycinin) of 60 kDa; Ara h 4, a Cupin (Legumin-type, 11S, Glycinin) of 37 kDa; Ara h 5, a Profilin of 15 kDa; Ara h 6, a (2S albumin) of 15 kDa; Ara h 7, a Conglutin (2S albumin) of 15 kDa; Ara h 8, a Pathogenesis-related protein, PR-10 of 17 kDa; Ara h 9, a Nonspecific lipid-transfer protein 1 of 9.8 kDa; Ara h 10, a 16 kDa oleosin, and Ara h 11, a 14 kDa oleosin.
Allergy to cats is extremely common, occurring in up to 25% of people with allergies. Cat allergy is more common than allergy to dog dander, which may be related to the potency of cat hair and dander as an allergen as well as the fact that cats are not generally bathed. Cat allergen is produced in large amounts, particularly by male non-neutered cats as the allergen is partially under hormonal control. Dander is constantly airborne, sticky, and found in public places, even where there are no cats. This is due to the dander being carried on the clothing of people who have cats, then shed in public places. Therefore, cat allergen is a component of house dust, even in homes where a cat has never lived. The size of the cat dander particles is extremely small, and is inhaled deep into the lungs. Cat dander is therefore a common cause of allergic asthma, and cat owners who are allergic to cats are more prone to the development of asthma symptoms(8, 9).
Major cat and dog allergens can be found in hair/dander extracts and saliva and are hence considered to be epithelial allergens. Eight different allergens have been identified in cat and many of them have been sequenced and cloned. Based on the IUIS websitei these allergens include: Fel d 1 a Uteroglobin of 14 and 4 kDa; Fel d 2 a Albumin of 69 kDa; Fel d 3 a Cystatin of 11 kDa; Fel d 4, a Lipocalin of 22 kDa; Fel d 5, a Immunoglobulin A of 400 kDa; Fel d 6, a Immunoglobulin M of 800-1000 kDa; Fel d 7, a von Ebner gland protein of 17.5 kDa; Fel d 8 a Latherin-like protein of 24 kDa. The major cat allergen, Fel d 1, has been characterized extensively by protein and immunochemical techniques and was recently expressed as a recombinant allergen. Fel d 1 represents an approximately 36 kDa dimer, which is composed of two 17 kd subunits(10).
Grass allergy is one of the most common and prevalent form of allergy that affects people with histories of it during certain seasons. It is present in the air in the late spring and early summer months, which can cause allergic rhinitis, allergic conjunctivitis and asthma. Direct skin contact with grass, from sitting in the grass or mowing the lawn, can cause itching of the skin, urticaria and atopic dermatitis. One of the most representative species is Phleum pratense, selected as leader of the grasses group. Nine different allergens have been identified of the species Phleum pratense. Based on the Allergen website′ these allergens include: Phl p 1, a Beta-expansin of 27 kDa; Phl p 2, a Grass group II/III of 10-12 kDa; Phl p 4, a protein of 55 kDa, Phl p 5 of 32 kDa, Phl p 6 of 11 kDa, Phl p 7 a calcium binding protein of 6 kDa, Phl p 11, Ole e 1-related protein of 20 kDa, Phl p 12 a profilin of 14 kDa and Phl p 13, polygalacturonase of 55 kDa.
Phragmites is a genus belonging to the group of grasses. Several species have been described including P. australis, or P. communis. Phragmites communis pollen has been reported to be allergenic in different areas. Pollination occurs between summer and fall depending on latitude and elevation.
Five different proteins with IgE binding capacity have been identified in genus Phragmites. Based on the Allergome websitei these allergens include: An expansin of 30 kDa; a protein belonging to the group 4 of grasses of 60 kDa; a ribonuclease of 35 kDa, a profilun of 14 kDa and finally a polygalacturonase.
Ragweed (Ambrosia) are weeds that growth mainly in central Europe. A plant lives only a season but the plant produces up to thousand of pollen grains. Warmth, humidity and breeze after sunrise help to release the pollen grains. Until now three different species have been related with allergy symptoms (Amborsia artemisiifolia (short ragweed), A. psilostachya (Western ragweed), and A. trifida (Giant ragweed)). Ten different allergens have been identified in short ragweed and many of them have been sequenced and cloned. In the case of Amborsia artemisiifolia, these are termed Amb a 1 to Amb a 10 according to the international nomenclature for allergens. Based on the IUIS websitei these allergens include: Amb a 1 a Pectate lyase of 38 kDa; Amb a 2 a Pectate lyase of 38 kDa; Amb a 3 a Plastocyanine of 11 kDa; Amb a 4, a Defensin like protein of 30 kDa; Amb a 5, of 5 kDa; Amb a 6, a lipid-transfer protein of 10 kDa; Amb a 7 a Plastocyanin of 12 kDa; Amb a 8 a Profilin of 14 kDa; Amb a 9 a Polcancin of 10 kDa and Amb a 10, a Polcalcin like protein of 18 kDa. For A. psilostachya only the allergen Amb p 5 has been described with unknown biological function. Only one allergen of 5 kDa has been also described in A. trifida. 
Weeds can be divided into homologous groups according to their classified allergenic extracts. Ambrosia was selected as one of the leaders of this group of plants. For that reason, results obtained with this pollen extract can be extrapolated to other weedsi.
Allergy can be treated by a number of known methods including allergen immunotherapy, specific immunotherapy (SIT), or Specific allergy vaccination (SAV) is a form of immunotherapy for allergic disorders in which the patient is vaccinated with increasingly larger doses of an allergen extract with the aim of inducing immunological tolerance. Allergen immunotherapy modulates the immune response to the allergen rather than ameliorating the symptoms induced by an allergic reaction, and can either reduce the need for medication, reduce the severity of symptoms or eliminate hypersensitivity altogether.
Although there is ample evidence that allergen immunotherapy is the only means, apart from allergen avoidance, to causally treat IgE-mediated allergic disorders caused by inhaled allergens and by stinging insects of the Hymenoptera group, immunotherapy with allergen extracts is not typically used for food allergy treatment. Only two recent studies have demonstrated a moderate clinical efficacy using sublingual immunotherapy in hazelnut and peach sensitized individuals respectively(3, 4).
In previous studies attempts have been made to induce a low-dose tolerance by feeding children with miniscule peanut traces which gradually become larger and larger in order to build up the immune system(4, 6). Although early clinical trials data indicates that peanut allergy can be ameliorated using immunotherapy(7), there is currently no confirmed treatment to prevent or cure allergic reactions to peanuts, with the only effective option for atopic individuals being to avoid foods that contain or are contaminated with whole peanuts, peanut particles and peanut oils and providing ready access to self-injectable epinephrine.
One of the risks of immunotherapy is that injection of an allergen to a sensitised patient can cause a severe allergic reaction or anaphylaxis. Since its first use in the beginning of the 20th century, many efforts have been made to further improve the safety and efficacy of allergen immunotherapy. One approach is to employ allergen vaccines with reduced allergenicity but with maintenance of immunogenicity.
U.S. Pat. No. 5,770,698 and EP0662080 disclose a process for removal of substances and other low molecular weight material in order to purify the allergen extract and to increase the final allergen/protein content. The process consists of disrupting the electrostatic, hydrophobic or other physical forces under such conditions as to disadhere non-allergenic compounds from the allergenically active proteins. The process can consist of a mild acid treatment by lowering the pH below the pI of the respective allergen proteins.
One of the various ways of reducing allergenicity consists of chemically modifying native allergen extracts with aldehyde, mainly formaldehyde and glutaraldehyde, to produce allergoids. This aldehyde treatment leads to reaction products (mainly polymers), which have lost part of their allergenicity (i.e. exhibit a reduction of IgE reactive B-cell epitopes), reducing allergic side-effects. At the same time, the native immunogenicity of the allergen is retained owing to unchanged T-cell epitopes. This route of allergen modification has been chosen by some manufacturers of allergen vaccines to develop commercially available products based on this principle. In general, there is a trend to further purify allergen extracts, carefully selecting the most important and clinically relevant allergens.
EP1834649 and EP1834648 disclose methods for producing allergen extracts; however such methods do not sufficiently remove contaminating low molecular weight proteins.
There is a need to further improve the safety and efficacy of medicaments for use in the immunotherapy of allergic disorders by optimising the allergen purification process to ensure that contaminating low molecular weight proteins, irritants and toxic components are eliminated.